title: Zinc finger nuclease mediated knockout of ADP-dependent glucokinase in cancer cell lines: effects on cell survival and mitochondrial oxidative metabolism.
abstract: Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.
authors.affiliation: Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand.
authors.forename: Susan
authors.initials: S
authors.lastname: Richter
authors.forename: Shona
authors.initials: S
authors.lastname: Morrison
authors.forename: Tim
authors.initials: T
authors.lastname: Connor
authors.forename: Jiechuang
authors.initials: J
authors.lastname: Su
authors.forename: Cristin G
authors.initials: CG
authors.lastname: Print
authors.forename: Ron S
authors.initials: RS
authors.lastname: Ronimus
authors.forename: Sean L
authors.initials: SL
authors.lastname: McGee
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
mesh_terms: D000818:Animals; D001483:Base Sequence; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002470:Cell Survival; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D005260:Female; D016368:Frameshift Mutation; D018628:Gene Dosage; D055786:Gene Knockout Techniques; D005818:Genetic Engineering / Q000379:methods; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism; D006019:Glycolysis; D045325:HCT116 Cells; D006593:Hexokinase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D010085:Oxidative Phosphorylation; D010101:Oxygen Consumption; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome; D047368:Tumor Burden
chemical_list: C486497:PPARGC1A protein, human; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D014157:Transcription Factors; C420842:ADP-dependent glucokinase; D006593:Hexokinase; D005941:Glucokinase; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific